Method and device for dosing small volumes of liquid

ABSTRACT

The invention relates to a method for dosing a volume of liquid (VF) of less than 1 !l that is to be dispensed, by means of a dosing pump, in particular a pipette ( 2 ). According to said method, a tip ( 13 ) is completely filled with a liquid and contains the liquid ( 16 ) to be dispensed at least in the vicinity of its outlet ( 13   a ), a volume of gas (VL) is drawn into the tip ( 13 ) via the outlet ( 13   a ) and a volume corresponding to the sum of the volume of liquid (VF) to be dispensed and the volume of gas (VL) is then supplied to the tip ( 13 ) in such a way that the volume of liquid (VF) is dispensed via the outlet ( 13   a ) in a contactless manner. The device comprises a plurality of dosing pumps that can be simultaneously actuated, in particular pipettes ( 2 ), which can be actuated by an injection plunger ( 6 ), in addition to a motor ( 1 ), which drives the injection plungers ( 6 ) and also a valve ( 3 ), which is located between the dosing pump and its respective tip ( 13 ) in order to fill each system completely with liquid without any gas.

The invention relates to a method for metering small volumes of liquid,and to a device for metering small volumes of liquid.

BACKGROUND

Increasing miniaturisation and parallelisation of biochemical,cellular-biological or molecular-biological test methods results in anincreased need for devices being able to dispense in a contact-free andhighly parallelized manner very small liquid volumes in the region ofless than 1 μl. Droplets of a few microlitres cannot be dispensed in acontact-free manner by conventional pipettes, but need to be detachedfrom the pipette by way of contacting a vessel surface or a liquidsurface. This however involves the danger of an undesired contaminationof the sample to be transferred.

For reproducible and exact pipetting of small liquid volumes it is knownto use a device being filled with an incompressible liquid in anair-free manner, and comprising a pipette cylinder and a plungerdisplaceably mounted therein. Cylinder and plunger are designed for theexact metering of the liquid volume to be dispensed via positivedisplacement of the plunger. In order to be able to dispense liquidvolumes of less than 1 μl in a contact-free manner using such devices,further auxiliary measures are necessary in order to overcome theadhesion forces which act between the liquid drop to be dispensed andthe remaining liquid column.

In order to overcome the named adhesion forces, it is known to useso-called piezo-pipettes comprising a specially designed nozzle and apiezo-crystal which acts on the nozzle in a manner, such that the liquiddrop is pressed out of the nozzle by way of a piezoelectric impulse.This impulse makes it possible to dispense very small liquid drops in acontact-free manner. The disadvantages of the piezo-pipette are its highcost and its not being suitable for highly-parallelized arrangementsand, furthermore, its not permitting the use of disposable pipette tips.

The publication EP-0876219 discloses a further way for overcoming thenamed adhesion forces. The pipetting device according to thispublication comprises an impulse generator which is arranged between thesyringe (plunger, pipette cylinder) and the tip and which effects animpulse on the liquid column located in the pipetting device. Onedisadvantage of this pipetting device is the fact that it is littlesuitable for highly parallelized arrangements, because for safe,highly-parallelized dispensing, it would need a multitude of impulsegenerators which would render the pipetting device correspondinglyexpensive. A further disadvantage is the fact that an additional deviceis required for filling the pipette device in an air-free manner.

It is this desirable to create a method as well as a device for meteringsmall liquid volumes of less than 1 μm, wherein method and device are tobe inexpensive and, in particular, are to permit reliable dispensing ofliquid volumes of even less than 100 nl.

SUMMARY OF THE INVENTION

A method for metering and dispensing a volume of a size of less than 1μl of a liquid to be dispensed is described, the method using a meteringpump, in particular a pipette, wherein a tip is filled with a liquid andat least in the region of the tip's exit opening contains the liquid tobe dispensed, wherein a gas volume is drawn into the tip via its exitopening, and wherein thereafter a volume corresponding to the sum of theliquid volume to be dispensed and the drawn-up gas volume is supplied tothe tip in a manner such that the volume of liquid to be dispensed isdispensed via the exit opening in a contact-free manner.

The invention is based on the idea of using a syringe with motor-drivensyringe plunger for metering very small volume units in the range forexample between 10 nl and 1 μl, wherein firstly a gas or air volume isdrawn into a liquid-filled tip, and then the syringe plunger is movedwith a large acceleration in the opposite direction by a stroke, suchthat the displaced volume corresponds to the sum of the drawn-in airvolume and the liquid volume to be dispensed. The liquid volume is suchdispensed by being pressed out of the syringe or its tip respectivelywith an impact which is sufficient to overcome the adhesion forcesacting on the liquid volume to be dispensed.

The method according to the invention makes it possible to meter anddispense very small volumes (lower limit of up to 10 nl) of a liquid tobe dispensed in an accurate and reproducible manner, surprisingly evenwhen using a syringe with a conventional, motor-driven syringe plunger.Therein, the volume which is dispensed from the tip or is displaced bythe syringe plunger respectively consists of two part volumes, namelythe drawn-up and then dispensed gas volume and the dispensed liquidvolume. During dispensation of the gas volume the syringe plunger isable to be accelerated to a high plunger speed which is necessary forthe liquid volume to be accelerated to a flow speed or exit speedrespectively being required for overcoming the adhesion forces. Anelectric motor, preferably a commercially available, inexpensiveelectric motor is perfectly suitable as a motor-drive, although suchmotor has a certain intrinsic inertia which renders the effect of themethod according to the invention non-obvious. The intrinsic inertia ofthe electric motor as well as its limited acceleration capability is ofa minor significance in the method according to the invention since themotor-drive is accelerated during the discharges of the gas volume whichis not critical with regard to time or speed. The inertia of the liquidfilled syringe is also of a minor significance during the discharge ofthe gas volume. The stroke of the syringe plunger being required fordischarging the gas volume, and the time needed for such discharge maybe varied within a wide range by varying the size of the gas volumeand/or by choosing a suitable diameter of the tip and/or of the syringeplunger so that for example even with a relatively sluggish electricmotor it becomes possible to achieve the plunger speed required fordispensing the liquid volume in a contact-free manner.

A suitable motor-drive is in particular an electric motor such as astepper motor or a servo motor. However, other drives as for example ahydraulic or pneumatic motor may also be suitable. Of whatever naturethe motor-drive is, it is important that the volume to be dispensed canbe metered exactly and that, during the discharge of the air volume, themotor drive is able to accelerate the syringe plunger in a manner suchthat the syringe plunger has a sufficiently high speed when the liquidvolume is to be dispensed. During the dispensing of the liquid volume,the syringe plunger is to be braked to a standstill very quickly. Thisbraking may be effected by the motor-drive. Advantageously an additionalbrake (e.g. a disk brake) is provided, which additionally or alonebrakes the syringe plunger and/or the motor-drive.

The method according to the invention has the advantages that aninexpensive electric motor can be used for driving the syringe, and thata single electric motor can be used for driving a highly parallelizedarrangement of for example 96 or 384 syringes which are operated inparallel. The method according to the invention has the furtheradvantage that no additional device such as an impulse generator isrequired in order to effect a suitable acceleration of the liquid.

In order to keep the friction resistance of the syringe as low aspossible and still have good sealing, it is advantageous to not attachthe sealing element on the moving syringe plunger but on the stationarypipette cylinder. An annular sealing element being mounted in thepipette cylinder in a stationary manner acts as seal between the pipettecylinder and the syringe plunger. The sealing ring is thus stationaryand the plunger acts as displacer. The syringe plunger slides with a lowresistance over the sealing element so that the syringe plunger can bedisplaced in an easy-running manner. Furthermore, there in no need forthe bore of the pipette cylinder to have a precise form, which meansthat an inexpensive pipette cylinder can be used. Since only the annularsealing element is worn, the syringe can be operated for a long time andin an inexpensive manner.

DESCRIPTION OF THE DRAWING

The method according to the invention as well as the device according tothe invention are hereinafter described in detail by way of severalexemplary embodiments in connection with a drawing in several figures,wherein:

FIG. 1 is a schematic representation of a pipetting device;

FIG. 2 shows a tip filled with liquid;

FIG. 3 shows a tip which is partly filled with air;

FIG. 4 shows a tip from which the liquid volume VF is dispensed;

FIG. 5 is a speed/time diagram for the plunger of the plunger pipette;

FIG. 6 is a longitudinal section through a plunger pipette;

FIG. 7 shows a tip with an air bubble between the system liquid and theliquid to be dispensed (sample).

DETAILED DESCRIPTION

FIG. 1 schematically shows a highly parallelized arrangement of forexample 96 or 384 metering pumps 2 being arranged in parallel. Themetering pumps are designed as syringes 2, wherein only two of thesesyringes 2 are shown. The arrangement according to the invention couldhowever also comprise only a single syringe 2. Each syringe 2 comprisesa pipette cylinder 5 and a syringe plunger 6 being mounted therein andbeing moveable in moving directions Z. All syringe plungers 6 areconnected to a bar 8 via plunger rods 7. The bar 8 is connected to theelectric motor 1 via a spindle 1 a. The syringe plungers 6 are thusdisplaced to and fro in the moving directions Z by the electric motor 1.The pipette cylinders 5 are mounted in a carrier plate 3 a. The carrierplate 3 a together with a valve plate 3 b forms a valve 3. The valveplate 3 b which is displaceable horizontally comprises a channel system10 with channels 11 and 12. Furthermore, tips 13 with nozzle openings 13a are arranged in the valve plate 3 b. The tips 13 are positioned abovea plate 14 with cavities 15. The syringe system must capable of beingcompletely filled with a liquid 16 a, for example with water, withoutany inclusions of air. This is accomplished by the three-way valve 3,which connects the syringes with reagent supply channels 11 or removalchannels 12, the valve being arranged between the syringe 2 and the tip13.

Furthermore, it may be advantageous to arrange a brake 9 on the spindle1 a or on the motor 1 in order to bring the rotating motor 1 to astandstill as quickly as possible.

The device shown in FIG. 1 is operated as follows.

In a first step, the syringe system is filled completely with a liquid16 a, e.g. with a system liquid such as water. The syringe system isfilled in a manner such that no inclusions of air are present.

In a second step, the sample liquid 16 (liquid to be dispensed) is drawninto the pipette tip 13 by way of aspiration from a supply container sothat the tip 13 is at least partly filled with the liquid to bedispensed.

In a third step, an air volume VL is drawn into the tip 13 via thenozzle opening 13 a of the tip 13.

In a fourth step, a predetermined volume of the liquid to be dispensedis dispensed in a contact-free manner by moving and accelerating thesyringe plunger 6 downwards (opposite to direction for aspiration), suchdisplacing a volume which is equal to the drawn-in air volume VL and thepredetermined liquid volume to be dispensed. Thereafter, the syringeplunger 6 is stopped, and the tip 13 (as shown in FIG. 2), is againcompletely filled with the liquid 16.

After the above described preparation (first and second step) aplurality of further predetermined liquid volumes VF can be dispensedvia the syringe 2 in a contact-free manner by repeating the third andfourth step. FIG. 2 is a schematic longitudinal section through a tip 13filled with liquid 16, wherein the liquid volume VF to be dispensed isshown in the region of the nozzle opening 13 a. This liquid volume VFforms part of the liquid 16 and in FIG. 2 is shown with a differenthatching only for simplifying understanding. In the following step (asshown in FIG. 3), the air or gas volume VL is drawn up into the tip 13.The gas volume VL is advantageously a multiple of the liquid volume VFto be dispensed, e.g. ten times the volume VF. For example, for a volumeVF of 50 nl of the liquid to be dispensed, the gas or air volume VL maybe 500 nl. Thereafter, the syringe plunger 6 is moved downwards with alarge acceleration by the electric motor, wherein the syringe plunger 6up to its standstill is to displace a volume corresponding to the sum ofthe air volume VL and the liquid volume VF to be dispensed, so that, asis represented in FIG. 4, the liquid volume VF to be dispensed ispressed out of the tip 13. The diameter of the nozzle opening 13 a aswell as the exit speed of the liquid are selected such that adhesionforces are overcome and the liquid volume VF which for example has avolume in the region between 10 nl and 1 μl is released from the tip 13.The procedure represented in the FIGS. 2, 3 and 4 may be repeatedsuccessively several times, wherein the dispensed liquid volumes VF maybe of the same size or wherein the dispensed liquid volume VF may bevaried by varying the stroke of the syringe plunger 6.

The nozzle opening 13 a has for example a diameter of 0.2 mm, and inparticular a diameter between 0.1 mm and 0.3 mm. The exit speed of theliquid from the nozzle opening 13 a is for example in the region between2 and 20 m/s and in particular 5 m/s. The air volume VL is for example950 nl, and the liquid volume VF to be dispensed for example 50 nl. Theelectric stepper motor 1 is controlled such that the syringe plunger 6is displaced upwards in 950 steps of the stepper motor and thendownwards in 1000 steps. During the downward movement, the plunger isfirstly accelerated and then braked. The resulting acceleration path BSin the tip 13 is shown in FIG. 5.

FIG. 5 schematically and by way of example shows a speed-time diagram ofthe speed v of the syringe plunger 6 moved downwards in the movementdirection Z (FIG. 1). During this movement, firstly the air volume VLand subsequently the liquid volume VF to be dispensed is discharged viathe nozzle opening 13 a. The syringe plunger 6 is accelerated during thedischarge of the air volume VL to a speed vmax, and retains this speeduntil the movement of the syringe plunger 6 is braked. During thedispensing of the air volume VL the syringe plunger 6 covers thedistance SL, and during the dispensing of the liquid volume VF it coversthe distance SF which is shown hatched. Depending on the size of the airvolume VL and of the liquid volume VF to be dispensed, dispensing of theliquid volume VF already begins during the movement at a constant speedvmax, for example at the point in time T, or for example not until thepoint in time T1 at which the braking procedure begins. Preferablydispensing of the liquid volume VF begins before the point in time T1and lasts, as shown in FIG. 5 with the hatched area, from the point intime T to the standstill of the syringe plunger 6. Since the liquid isincompressible, during dispensing in such a way, the exit speed of aliquid element located in the exit opening 13 a is about proportional tothe speed of the syringe plunger 6, or acceleration of the liquidelement is proportional to the acceleration of the syringe plunger 6. Ifthe syringe plunger 6 for example has an inner diameter of 2 mm and theexit opening 13 a an inner diameter of 0.2 mm, then the speed of theliquid element at the exit opening 13 is about 100 times larger than thespeed of the syringe plunger 6.

In order to be able to achieve an as large as possible negativeacceleration during the braking of the syringe plunger 6, the stop rampSR must be suitably steep. Should the electric motor 1 not be able toproduce an adequately steep stop ramp SR, the steepness may be increasedby an additionally acting brake 9. In the exemplary embodiment of FIG. 5the stop ramp SR represents such a negative acceleration that the liquidelement located in the exit opening 13 a experiences a negativeacceleration of about 100 m/sec².

It is also evident from FIG. 5 that the air volume VL is required tohave such a size that the syringe plunger 6 is able to reach the maximumspeed vmax. If the air volume VL is selected too small, the syringeplunger 6 must already be braked before it has reached the maximum speedvmax. The maximum speed vmax is preferably matched to the cross sectionof the exit opening 13 a, to the liquid volume VF to be dispensed, andin particular also to the viscosity of the liquid to be dispensed. Thesize of the exit opening 13 a is preferably selected to be adapted tothe size of the liquid volume VF to be dispensed, wherein the geometryas well as the surface nature of the exit opening 13 a are to be takeninto account. The smaller the liquid volume VF to be dispensed, the morecareful the named parameters need to be selected. The smaller the liquidvolume VF to be dispensed is, the steeper the stop ramp SR should be inorder to ensure a secure, contact-free dispensing of the liquid volumeVF. The negative acceleration of the stop ramp preferably lies in such arange that a negative acceleration between 50 m/sec² and 200 m/sec² actson the liquid element located in the exit opening 13 a.

FIG. 6 shows a longitudinal section through an easy-running plungerpipette 2. A sealing ring 19 is rigidly connected to the pipettecylinder 5. The syringe plunger 6 is mounted to be moveable in themoving directions Z and to displace a volume within the pipette cylinder5.

FIG. 7 shows a longitudinal section through a tip 13, in which liquid islocated with an air bubble 18 between the sample liquid 16 and thesystem liquid 16 a. This air bubble 18 prevents mixing of the sampleliquid 6 with the system liquid 16 a. The size of the air bubble 18 ispreferably selected in dependence on the cross section of the tip 13.For example, for a liquid volume VF of 100 nl to be dispensed, the airbubble 18 may have a volume of about 1 μl. With very small liquidvolumes VF to be dispensed (e.g. 10 nl), preferably a very small airbubble 18 is selected, or the system is filled with liquid in a mannersuch that no air bubble 18 results between the sample liquid 16 and thesystem liquid 16 a, i.e. completely without inclusions of air or gas.

FIG. 1 further shows a control device 17 for controlling the meteringdevice according to the invention. This control device 17 may be anintegral part of the metering device, but may also be designed as anindependent device which is connected to an existing metering device.The control device 17 is designed for controlling the drive device 1 ofthe metering pump 2, such that the metering pump 2, directly before aliquid volume VF is dispensed, aspires a volume of system liquid 16 acorresponding to the gas volume VL and for dispensing the volume VF ofsample liquid subsequently delivers a liquid volume of system liquid 16a corresponding to the sum of the gas volume VL and the liquid volume VFto be dispensed, so that a liquid volume VF of the sample 16 isdispensed from the tip 13.

1-14. (canceled)
 15. A method for dispensing a volume of liquid, themethod for use with a metering pump and a pipette having a tip having anexit opening, the method comprising the steps of: filling the tip withliquid such that at least in the region of its exit opening it containsthe liquid to be dispensed; drawing a gas volume up into the tip via theexit opening; dispensing via the exit opening, a volume corresponding tothe sum of the volume of the liquid to be dispensed, said volume beingof a size less than 1 μl, and the gas volume.
 16. The method of claim15, characterized in that the gas volume is a multiple of the volume ofthe liquid to be dispensed.
 17. The method of claim 15, characterized inthat metering is repeated several times, wherein identical or differentliquid volumes are dispensed successively.
 18. The method of claim 15,characterized in that the pipette comprises a pipette cylinder and asyringe plunger moveably mounted therein, and further characterized inthat, for dispensing the volume of the liquid, the syringe plunger ismoved in a manner such that it displaces a volume corresponding to thesum of the volume of the liquid to be dispensed and the gas volume. 19.The method of claim 18, characterized in that, during the volumedisplacement, the syringe plunger is firstly accelerated to a maximumspeed and then, during the dispensing of the volume of the liquid to bedispensed, is braked to a standstill.
 20. The method of claim 19,characterized in that a liquid element located in the exit opening isbraked with a negative acceleration of at least 50 m/sec².
 21. Themethod of claim 20 in which the negative acceleration is about 100m/sec².
 22. The method of claim 15, characterized in that the liquidvolume is dispensed via the exit opening with a throughput speed of atleast 3 m/s.
 23. The method of claim 15, characterized in that theliquid volume to be dispensed has a size in the region between 10 nl and1 μl and is dispensed in a contact-free manner.
 24. An apparatuscomprising: a metering pump; a pipette; the pipette having a tipconnected thereto in a liquid-conducting manner; the tip comprising anexit opening for dispensing the liquid volume; a drive device fordriving the metering pump, characterized in that the metering pump isable to be driven in a manner such that a suctioning is effected at theexit opening directly before dispensing the liquid volume, andthereafter a dispensing is effected, in order to first suction a gasvolume via the exit opening, and thereafter to dispense the gas volumeas well as a liquid volume via the exit opening; the liquid volume beingof a size of less than 1 μ.
 25. The apparatus of claim 24, characterizedin that the drive device comprises an electric motor.
 26. The apparatusof claim 24, characterized in that a valve is arranged between themetering pump and the tip, the valve being designed in a manner suchthat the liquid-conducting connection between the metering pump and thetip is able to be completely filled with liquid.
 27. The apparatus ofclaim 24, characterized in that the tip comprises an exit opening with adiameter between 0.1 mm and 0.3 mm.
 28. The apparatus of claim 27wherein the exit opening diameter is about 0.2 mm.
 29. The apparatus ofclaim 24, characterized in that the pipette comprises a pipette cylinderwith an annular sealing element being mounted in a stationary mannertherein and being designed as a seal between the pipette cylinder and asyringe plunger being movably arranged in the pipette cylinder.
 30. Theapparatus of claim 24, further comprising a control device, the controldevice characterized in that the control device is designed forcontrolling the drive device of the metering pump in a manner such, thatthe metering pump, directly before dispensing the volume of liquid to bedispensed, displaces a liquid volume corresponding to the gas volumeaway from the tip, and thereafter displaces a liquid volumecorresponding to the sum of the gas volume and the volume of the liquidto be displaced towards the tip.